(searched for: Assessment of the Coagulase Test in the Identification of Staphylococcus Aureus Strains)
International Journal of Microbiology, Volume 2020, pp 1-6; doi:10.1155/2020/6512106
Staphylococcus spp. is most often implicated in nosocomial infections. The objective of this study is to evaluate the susceptibility to antibiotics and the biofilm formation capacity of staphylococci species isolated from surfaces and medicotechnical materials at the university hospital center of Abomey-Calavi/Sô-Ava in Benin. Samples were collected according to ISO/DIS14698-1 standard from the surfaces and medicotechnical materials by the dry swab method. The isolation of Staphylococcus strains was performed on Chapman agar, and their identification was performed using microscopic and biochemical methods. The susceptibility of Staphylococcus isolates to antibiotics was evaluated by the disc diffusion method according to EUCAST and CLSI recommendations. The biofilm formation was qualitatively assessed using microplates. Of the 128 surfaces and medicotechnical material samples analyzed, 77% were contaminated with Staphylococcus spp. Thirteen species of Staphylococcus were isolated in different proportions but the pediatric department was the most contaminated (33%) by S. aureus. Resistance to antibiotics considerably varies according to the species of Staphylococcus. However, antibiotics such as chloramphenicol and vancomycin are the most effective on S. aureus, whereas coagulase-negative staphylococci developed less resistance to gentamycin and ciprofloxacin. The biofilm test reveals that 37% of our isolated strains were biofilm formers. Although regular monitoring of hospital hygiene is crucial, the optimal use of antibiotics is a cornerstone of reducing antimicrobial resistance.
Frontiers in Veterinary Science, Volume 7; doi:10.3389/fvets.2020.00376
Canine superficial pyoderma (CSP) is a bacterial infection secondary to several skin diseases of the dog. Staphylococcus pseudintermedius, which is a commensal bacterium of the dog's skin, is the leading agent found in dogs affected by CSP, which can progress to deep pyoderma. It is also of clinical significance because S. pseudintermedius strains carry antimicrobial resistance genes, mainly the mecA gene. In this descriptive longitudinal study, molecular characterization of bacterial isolates from dogs affected by CSP was performed in addition to phenotyping, antimicrobial profiling, and assessment of resistance carriage status. Fifty dogs (24 females and 26 males) attending the CES University Veterinary Teaching Hospital were included in the study. CSP was confirmed according to clinical signs and cytological examination. Swabs were taken from active skin lesions for bacterial culture, and phenotyping and antimicrobial resistance profiles were assessed using API-Staph phenotyping and the Kirby–Bauer method, respectively. We also performed molecular detection and characterization of the mecA and nuc encoding gene of coagulase-positive Staphylococci. The mecA gene frequency was established by qPCR amplification of a 131bp gene fragment. Data were evaluated by descriptive statistics. Erythema, peeling, pruritus, and alopecia were the predominant symptoms (72, 56, and 46%, respectively). We isolated bacteria compatible with Staphylococcus species from all samples tested. API phenotyping showed 83.1 to 97.8% compatibility with S. pseudintermedius. PCR-genotyping resulted in 15, 3, and 1 isolates positive for S. pseudintermedius, S. aureus, and S. schleiferi, respectively. Isolated strains showed high susceptibility to Imipenem, Ampicillin/Sulbactam, and Rifampicin (100, 94, and 92%, respectively). The highest resistance was against Vancomycin and Trimethoprim/Sulfamethoxazole (98 and 74%, respectively). S. pseudintermedius, S. aureus, and S. schleiferi isolates were cloned and shared 96% sequence homology. Finally, we found 62% carriage status of the mecA gene in isolates of CSP patients, although only 36% of the isolates were methicillin-resistant. Identification of three Staphylococcus species causing CSP, high-level resistance against conventional antimicrobials, and carriage of the mecA gene highlight the importance of performing molecular characterization of bacteria causing dermatological conditions in dogs.
Journal of Spectroscopy, Volume 2016, pp 1-9; doi:10.1155/2016/5436821
Optics based technologies are being advanced by many diagnostic companies around the globe. This resurgence is being driven by several factors including novel materials, enhanced computer power, nonlinear optics, and advances in algorithmic and statistical analysis. This study expands on a previous paper that evaluated the capability of a reagent-free optical profiling platform technology that used multiwavelength transmission spectroscopy to identify bacterial pathogens from pure culture. This study combines multiwavelength angular scattering with transmission based analysis into a single algorithm that will identify bacterial pathogens. Six predominant organisms, S. aureus, E. coli, K. pneumoniae and P. aeruginosa, E. faecalis, and coagulase negative Staphylococcus, were analyzed from a total of 753 clinical isolates received from three large community hospital systems. The bacterial identification method used for comparison in this study was the Vitek-2 (bioMerieux) which utilizes a biochemically based identification system. All of the clinical isolates received were blinded as to their identification until completion of the optical analysis. Sensitivities ranged from 87.7 to 94.6% with specificities ranging from 97.2 to 99.9% indicating that optical profiling is a powerful and exciting new technology that could be developed for the rapid identification of pathogens without the use of chemical reagents.1. IntroductionAccurate and rapid bacterial identification is essential for correct disease diagnosis, treatment of infection, and tracing back of disease outbreaks associated with microbial infections. Traditionally, clinical methods for bacterial identification have relied on phenotypic identification of the causative organism using Gram staining, cultural growth characteristics, and biochemical reaction based techniques. These methods of bacterial identification, while in broad use today, suffer from a major drawback as certain strains exhibit nontraditional and unique biochemical characteristics that do not fit into the classical patterns that have been established to characterize a particular genus and species. Further, phenotypic properties can be unstable at times and expression can be dependent upon changes in environmental conditions, for example, growth substrate, temperature, and pH. Despite these limitations, early advances designed to produce more rapid microbial detection focused on the automation of these phenotypic identification methods .Alternatives to the phenotypic methods for bacterial identification available today include a variety of spectroscopy and spectrometry based methodologies in various stages of investigation and development [2–14]. Many of these systems require less than 6 hours for specimen analysis, can analyze whole bacterial cells , are reproducible over a broad mass range, and are often specific enough to identify antibiotic-resistant bacteria [12, 13]. Despite this renewed interest in spectroscopic analysis, there exists a persistent focus on the use of either the scattering properties of the cells [9, 15, 16] or the absorption properties of the cellular constituents [3, 14]. Neither of these approaches in isolation has been successfully brought to market for bacterial identification. The combination of multiwavelength transmission and angular scattering measurements shown in this study is unique to the SpectraWave technology.SpectraWave is being developed as a low-cost, optical profiling platform for the characterization of particulates which in this case are the bacterial pathogens of interest. At current stage of development, it integrates multiwavelength transmission and angular scattering measurements to ascertain the physical, structural, and compositional characteristics of bacterial cells. In this study, the performance of SpectraWave for bacterial identification was assessed using Vitek-2 (bioMerieux), a biochemically based bacterial identification system, as a reference system. The characteristics of a generic matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry system is also presented for comparison in Table 1. The techniques differ significantly in three main areas, namely, the assay format, a priori testing requirements, and the time to results (Table 1).Table 1: Comparison of SpectraWave (Claro Scientific) and Vitek-2 (bioMerieux) bacterial identification techniques.With regard to the assay format, SpectraWave is an optically based method which uses no chemical reagent for either sample preparation or analysis. All biochemically based systems currently on the market require the laboratory personnel to perform a Gram stain prior to initiating the bacterial identification procedure. This information is used to select the correct reagent card (Gram positive or Gram negative) that will be used for analysis in the Vitek-2. This step is not necessary for optical profiling as no a priori information is needed in the identification algorithm. Lastly, optical profiling can provide identification within minutes while results from the biochemically based systems are available in approximately 4–10 hours. Although manual sample preparation was used during this study, bacterial identification using SpectraWave was completed in less than 15 minutes. When fully automated, the time to identification will be less than 5 minutes. This compares very favorably to methods employed in the clinical laboratory today.Optical profiling in its most basic sense encompasses the analysis of the interactions of light with matter. Previous studies have demonstrated that a rich body of quantitative information can be extracted from spectra when the combined contribution of the absorption and scattering properties are considered jointly as a function of wavelength [17–20]. This technique enables the detection, identification, and characterization of a variety of cells, pathogens, and disease markers using their optical signatures [19, 20]. Individual modes of interaction (absorption, scattering, angular scattering, etc.) have their respective advantages, yet they are restricted by the amount of information that can be extracted from the measurements. When combined, they supply a unique set of independent, complimentary, and confirmatory data that can be used to identify, characterize, and quantify a sample with high levels of sensitivity and specificity. Combining the simplicity of the spectroscopic measurement and reagentless nature of the technique, optical profiling has a great potential to be developed for rapid, low-cost, and easy to operate diagnostic devices.In this study SpectraWave was evaluated for the rapid and accurate identification of bacterial pathogens from pure culture. For this purpose, 753 clinical isolates of six bacterial pathogens (S. aureus, E. coli, K. pneumoniae and P. aeruginosa, E. faecalis, and coagulase negative Staphylococcus spp.) were received from three large local community hospitals. These six target species accounted for over 85% of the total number of organisms isolated from positive blood cultures in participating hospitals during the study period. All samples were analyzed in a blinded fashion.2. Materials and Methods2.1. Sample PreparationBacterial isolates from positive blood cultures that had been subcultured onto either MacConkey and/or blood agar were numbered sequentially and packaged at the participating community hospitals and shipped via courier to Claro Scientific on a twice weekly basis. Upon receipt, the samples were stored at room temperature until assayed. Each shipment contained a sealed envelope with the identification of each isolate as determined by the Vitek-2 (bioMerieux) by hospital laboratory personnel.All culture plates were visually inspected prior to use for obvious contamination events. All plates that were deemed contaminated were excluded from the study. A single colony was selected from each acceptable culture plate and put in Erlenmeyer flasks containing 50 mL of sterile tryptic soy broth (BD Franklin Lakes, NJ). All samples were grown to stationary phase (19–24 hrs at 37° ± 2°C) using standard techniques. Samples were prepared for spectroscopic analysis by collection of 1 mL aliquots from the stationary phase culture transferred to 1.5 mL sterile Eppendorf tubes. Samples were centrifuged for three minutes at 13,000 RPM (ThermoFisher Accuspin Microfuge 12). Tubes were removed from the Microfuge and the supernatant was slowly drawn off and discarded. The remaining pellets were resuspended in deionized water and vortexed until homogeneous distribution of cells in the suspension. The washing process (centrifugation, aspiration, and resuspension) was repeated three times until the suspensions were clear of the growth media.2.2. Transmission MeasurementAppropriate aliquots of the final cell suspensions were diluted into 3 mL of deionized water in a 1 cm path length quartz cuvette. The degree of dilution for each sample was selected to yield maximal optical density values between 0.4 and 0.8 absorbance units (AU) in the wavelength range of 190–220 nm. The transmission measurements were collected using diode array spectrometers. The following diode array spectrometers were tested and found to be equivalent for pathogen identification: Agilent 8453 (Santa Clara, California), Ocean Optics HR4000, and Ocean Optics HR2000 (Dunedin, Florida). The data reported herein were collected with the Agilent 8453. Other commercial spectrophotometers can be used provided that the spectrophotometers have a minimum of 1 nm wavelength resolution, a high signal-to noise ratio (>99.9%), and an acceptance angle smaller than 2°. All transmission spectra were visually inspected and removed from the data set prior to any analysis if they exhibited aberrant absorption and/or scattering signal in the visible-IR wavelength region due to manual sample preparation errors, diffraction artifacts, and so forth.2.3. Ang
Published: 19 March 2013
Visnyk of Dnipropetrovsk University. Biology, medicine, Volume 4, pp 23-29; doi:10.15421/021304
The paper presents monitoring results of the spread of opportunistic and pathogenic microorganisms in patients of surgical departments of the Dniprodzerzhynsk city hospital No 7. 1464 strains of bacteria isolated from biological material of the patients from January to December 2012 were studied. Relevant standard methods of research and data interpretation in accordance with the regulatory guidelines were used. The microorganisms’ sensitivity to antibiotics was determined by the disk diffusion method. Assessment of the resistance of isolated microorganisms to antibiotics was made with the software Whonet 5.1. At the first stage of investigation sampling biological material and inoculation in the culture medium were made. The discharges of wounds, throat, nose, ears, vagina and urethra, and also urine from patients of surgical departments were sampled for bacteriological analysis. The main substratum was 5% blood agar. There may additionally be used the selective growth media (yolk-salt agar, Endo, and Saburo). At the second stage we identify microorganisms with bacterioscopic, bacteriological and biochemical methods. Identifying microorganisms of the genus Staphylococcus was made by the reaction of lecithinase presence, plasma-coagulation reaction and the mannitol oxidation reaction. For the identification of bacteria of the family Streptococcaceae the growth pattern in 0.5% sugar medium was used. It was differentiated from bacteria of the genus Enterococcus by plating onto egg yolk agar base and milk with 0.1% methylene blue. Identification of bacteria of the Enterobacteriaceae family was made by studying their colonies on dense differential diagnostic media. Suspicious colonies were transferred on a combined medium for primary identification (Olkenitsky's medium). Then the biochemical signs of enterobacteria were studied in the minimum number of tests. The third phase of the study included the determination of the sensitivity of microorganisms to antibiotics. To do that, we use the disc diffusion method. The antibiogram tests of isolated bacterial strains used Mueller-Hinton agar. According to the data obtained we assign of microorganisms to certain category of sensitivity: sensitive, moderately resistant or resistant. Monitoring of prevalent microorganisms showed that 61% (893 of 1464) isolates were gram-positive bacteria, 696 strains of them are staphylococci. 477 of these are identified as S. aureus, representing 68.5% of all Staphylococcus. We found coagulase negative staphylococci in 192 patients, and the strains of S. haemolyticus are dominated. Strains of the family Streptococcaceae isolated from 197 patients. Among them the E. faecalis accounted for 66% of the total number of Streptococcus. Gram negative bacteria were presented by the families Enterobacteriaceae and Pseudomonaceae. Enterobacteriaceae accounted for 90.7% (518 of 571). E. coli plays the leading role and accounted for 42% of all Enterobacteriaceae. The strains of P. aeruginosa were identified in 53 patients. S. haemolyticus has played an important role as a pathogen as well as S. aureus. Its resistance to antibiotics is much higher than that of S. aureus. Although S. haemolyticus is opportunistic coagulase negative, it can be isolated from patients not only with chronic, but with acute infection. Thus nowadays the strains of S. haemolyticus gained high pathogenic and virulent properties.
Journal of Dairy Research, Volume 80, pp 223-226; doi:10.1017/s0022029913000022
The publisher has not yet granted permission to display this abstract.
BMC Infectious Diseases, Volume 12, pp 286-286; doi:10.1186/1471-2334-12-286
The staphylococci are implicated in a variety of human infections; however, many clinical microbiology laboratories in Nigeria do not identify staphylococci (in particular coagulase negative staphylococci - CNS) to the species level. Moreover, data from multi-centre assessment on antibiotic resistance and epidemiology of the staphylococci are not available in Nigeria. This study investigated 91 non-duplicate staphylococcal isolates obtained from the microbiology laboratories of eight hospitals in Nigeria during the period January to April 2010. Identification and antibiotic susceptibility testing was performed using the VITEK 2 system, detection of resistance genes by PCR, and molecular characterization was determined by SCCmec typing, spa and multilocus sequence typing (MLST). All the isolates were susceptible to mupirocin, tigecycline, vancomycin and linezolid, but 72.5% of CNS and 82.3% of Staphylococcus aureus were resistant to cotrimoxazole, while multiresistance was observed in 37 of the 40 CNS isolates. Untypeable SCCmec types (ccrC/Class A mec and ccr-negative/Class C2 mec gene complex) in two methicillin-resistant S. aureus (MRSA) were identified. Additionally, ccr-negative/Class A mec and ccr type 4/Class C2 mec gene complex was detected in one isolate each of S. sciuri and S. haemolyticus, respectively. The S. aureus isolates were classified into 21 spa types including two new types (t8987, t9008) among the methicillin-susceptible S. aureus (MSSA) isolates. Two (CC8-SCCmecnon-typeable and CC88-SCCmec IV) and four (CC8-SCCmec III/IV/V; CC30-SCCmec II/III; CC88-SCCmec IV; and ST152-SCCmecnon-typeable) MRSA clones were identified in Maiduguri (North-East Nigeria) and South-West Nigeria, respectively. The proportion of Panton-Valentine leukocidin (PVL)-positive MSSA was high (44.4%) and 56.3% of these strains were associated with sequence type (ST) 152. The identification of multiresistant mecA positive S. haemolyticus and S. sciuri from clinical samples indicates that characterization of CNS is important in providing information on their diversity and importance in Nigeria. There is the need to develop new SCCmec classification methods for non-typeable methicillin-resistant staphylococci, and to curtail the spread and establishment of the S. aureus ST152 clone in Nigeria. The study presents the first report of a PVL-positive ST152-SCCmecnontypeable MRSA and SCCmec typing of methicillin-resistant CNS in Nigeria.
Published: 1 January 2008
Journal of Venomous Animals and Toxins including Tropical Diseases, Volume 14, pp 294-302; doi:10.1590/s1678-91992008000200007
Endophytic microorganisms are relatively unstudied as potential sources of novel natural products for medical and commercial exploitation. The aim of this work was to investigate some Brazilian tropical savannah trees Cassia leptophylla and Prunus spp. in order to isolate the endophytic microorganisms associated with these plants. The samples were disinfected to eliminate the epiphytic population. Colonies were diluted and displayed as drops in media and growing colonies were inactivated. Staphylococcus coagulase-positive strain was used as indicator microorganism and subjected to the antibioses test. Data showed that the microorganisms isolated from Cassia leptophylla had no inhibition against Staphylococcus . On the other hand, microorganisms isolated from Prunus spp. leaves showed antibacterial activity and inhibited Staphylococcus when cultivated in peptone agar as well as in yeast extract agar. Investigation proceeds in order to classify the isolated microorganisms presenting bioactive substance and exploit the potential of the compounds produced to inhibit the indicator bacteria. Other bioactive properties will be investigated. Key Words: endophytic microorganisms, plants, antibacterial activity, bioactive compounds. INTRODUCTION The need for new and useful compounds to provide assistance and relief in all aspects of human condition is ever-growing. Drug resistance in bacteria, the appearance of life-threatening viruses, and the increase in the incidence of fungal infections in the world's population all underscore our inadequacy to cope with these medical problems. Endophytic microorganisms that colonize internal tissues of plants without causing any negative effects are relatively unstudied as potential sources of novel natural products for medical and commercial exploitation. Some endemic plants having an unusual longevity can generate bioactive natural products associated with endophytic microorganisms that produce the same natural products (1). Presumably, the simplest biological arrangement between these organisms is that the plant provides nutrition for the microbe and this one provides some form of protection for the plant. Endophytes promote plant growth and yield, suppress pathogens, may help to remove contaminants, solubilize phosphate, or contribute to assimilate nitrogen to plants (12). Brazilian tropical savannah plants are likely to be excellent specimens to begin a search for endophytic microorganisms (11, 15). In Brazilian tropical savannah soils with native plants, the natural populations of actinomycetes in the microbiota can be superior to 75%, with predominance of the genera Streptomyces (6, 8, 16). As a group, the Streptomycetes provide nearly 80% of all of the world's antibiotics produced (3, 4). A search reveals that most of these organisms have their origins in soil (10), and another biologically important niche that has been overlooked as a source of novel are the tissues of higher plants. This specialized and unique biological niche that supports the growth of microbes is the intracellular space between cells of higher plants. Novel endophytes usually have associated with them novel secondary natural products (17). The aim of this work was to investigate the Brazilian tropical savannah trees Cassia leptophylla and Prunus spp. in order to isolate the associated endophytic microorganisms with potential for producing antibacterial substances. MATERIALS AND METHODS The Brazilian tropical savannah plants Cassia leptophylla and Prunus spp. were collected in an ecological reserve at São Carlos, São Paulo State, Brazil, and analyzed at the Microbiology Laboratory, at Federal University of São Carlos. To eliminate the epiphytic population, the plants (leaves and caulis) were washed with distilled sterile water and neutral detergent and its surface disinfected with ethanol (70%/1min), sodium hypochlorite (2%/6min) and ethanol (70%/30s) and consecutively rinsed with distilled sterile water (10). The surface-disinfected plants were then aseptically sectioned into 0.5+/-0.7cm fragments, distributed onto the isolation media, yeast extract agar (YE) and peptone agar (PA), and incubated at room temperature for 4 days. At the same time, the disinfected plants were homogenized with peptone water 0.1% (1:9). Decimal dilutions were made and 100µl aliquots were displayed on the surface of agar medium and incubated (room temperature/4 days). From each of these plates, some colonies representing different morphologies were then picked. Preliminary microbial identification using Gram and Ziehl-Neelsen staining, melanin production and catalase activity were done. Selected colonies were diluted in peptone water (0.1%) and displayed as drops (Pasteur pipette) in PA and YE as an additional part. Petri dishes were incubated (room temperature, and 37°C/48h). The bioassays were conducted using growing colonies in PA and YE and inactivating them by chloroform (20min). Plates were opened (30min) to evaporate the substance. At the same time, the reactivation of Staphylococcus coagulase-positive strain (BHI broth 24h/37°C) was made and one aliquot was used as the indicator microorganism. 200µl of the culture properly reactivated was transferred to 10ml of semi solid BHI medium and shaken. This mixture was deposited onto the surface of plates containing the inactivated microorganisms and incubated (37°C/48h) for the observation of inhibition halos. RESULTS Ten colonies, chosen among those isolated from Cassia leptophylla and Prunus spp., were selected. Data showed that the microorganisms isolated from Cassia leptophylla had no antibiosis against Staphylococcus coagulase-positive strain when cultivated in PA and YE ( Figure 1 ). The macroscopic evaluations of one isolate from Prunus spp. were made. The observation of growing colonies in PA and YE were made by visual inspection of the morphological characteristics. The isolated microorganism showed different growing aspects when cultivated in these media (room temperature/48h). In YE, the colonies were yellowish, big, glowing and mucous, and in PA, white, small and with irregular borders ( Figure 2 ). Gram-positive bacilli and spores as well as bacilli with one or two apical spores in Gram staining were observed, suggesting that only one propagule was isolated. No alcohol-acid cells in Ziehl-Neelsen staining were observed. It was not observed the melanoid pigment production. The catalase activity was positive. The investigation continues in order to identify the isolated endophytic microorganism. The microorganisms isolated from Prunus spp. leaves showed distinct inhibition zones against Staphylococcus coagulase-positive strain. The white colony showed antibacterial activity against Staphylococcus , with inhibition zones of 1.6cm and 2.1cm diameter in media when cultivated in PA and YE, respectively ( Figure 3 ). Similarly, the inhibition of Staphylococcus by the other isolated microorganism (yellowish colony) presented inhibition zone of 2.0cm and 1.4cm diameter in media when cultivated in YE and PA, respectively ( Figure 4 ). DISCUSSION In Pernambuco state, Brazil, Brito (2) isolated actinomycetes from beans ( Phaseolus vulgaris ). They showed that 6% of the isolated microorganisms had antibiotic activity against Staphylococcus aureus and Bacillus subtilis . In this present study, the isolated microorganisms from Prunus spp. showed activity against the tested Staphylococcus coagulase-positive strain. Sardi et al . (14), working in Italy with about 500 endophytic actinomycetes isolated from roots from 28 different plants, showed that the Streptomyces were the predominant isolated genera. In Brazil, Matsuura (7) isolated 31 endophytic actinomycetes from roots and leaves from caupi beans ( Vigna unguiculata ), with special attention to Streptomyces followed by Streptosporangium and Nocardiopsis. According this author, 20% of the isolated microorganisms presented antimicrobial activity against Gram-positive bacteria and Candida albicans . Yoo et al . (18) analyzed the capability of the Streptomyces spp. CS684 in order to inactivate methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Data showed that the Streptomyces spp. CS684 presented antibacterial activity against MRSA and VRE. Gu et al . (5) analyzed the capability of Streptomyces roseosporus strain in the inhibition of Staphylococcus aureus and showed that the S. roseosporus strain exhibited potent antibacterial activity against S. aureus . Parvateesam and Bulchandani (9) showed that several strains of actinomycetes were isolated from soil samples collected from various localities of Ajmer District, India. These isolates were tested for their antagonistic properties against few test organisms like Bacillus subtilis , Escherichia coli , Pseudomonas aeruginosa , Staphylococcus spp., Saccharomyces cerevisiae and Aspergillus niger . Some of these actinomycetes strains exhibited antimicrobial activity against bacteria, but no antifungal activity was observed. A total of 90 different Streptomyces isolates were recovered from 36 soil samples and assessed for their antibacterial activity (13). The antibiotic activity against a wide range of bacteria was exhibited by 54% of the isolates which were effective against B. subtilis (57%), S. aureus (47%), E. coli (24%), Klebsiella spp. (16%), and Shigella spp. (12%). The lowest activity (8%) was exhibited against Pseudomonas spp. and Salmonella spp. The antibacterial activity of the isolates was divided into four groups according to the diameter of the produced inhibition zone. Groups 3 and 4 with larger inhibition zones indicated their potential as a possible source of novel antibiotics. In this respect, our results (2.14cm diameter media) probably are pointing to the isolation of an antibacterial compound. Data exhibited here suggests that Brazilian tropical savannah plants are good source to search endophytic microorgani
Brazilian Journal of Microbiology, Volume 35; doi:10.1590/s1517-83822004000100010
Staphylococcus spp are not usually isolated from the oral cavity, and when this occurs, they are considered to belong to the transitory microbiota. Individuals with periodontal disease represent possible reservoirs of these opportunistic bacteria in the oral cavity. The use of antibiotics for treatment of periodontal disease or other infections may predispose to the increase of the number of Staphylococcus spp. in the oral cavity. These microorganisms easily become resistant to antibiotics, and may result in superinfection. The purpose of this study was to evaluate the presence of Staphylococcus spp. in the oral cavity and in periodontal pockets of patients with chronic periodontitis, identify the isolates and verify the relationship between the presence of Staphylococcus spp. in the oral cavity and presence of periodontal pockets. The study included eighty-eight patients, 25-60 years of age, with chronic periodontitis, and at least two sites with probing depth > 5 mm. Individual data examination was assessed. Then, samples were colleted from the periodontal pocket with the aid of paper tips and from the oral cavity through mouth rinses. Out of the total of patients, 37.50% presented Staphylococcus spp. in the periodontal pocket and 61.36% in the oral cavity, 27.27% presented the bacteria in both sites. S. epidermidis was the most prevalent specie in the periodontal pocket (15.9%) and oral cavity (27.27%). The occurrence of higher proportions of nonresident's microorganisms in subgingival samples and oral sites may represent significant problem in causing and maintaining periodontal infections. Key words: Staphylococcus , opportunistic bacteria, periodontitis. RESUMO Staphylococcus spp. não são usualmente isolados a partir da cavidade bucal. Quando presentes, são considerados pertencentes à microbiota transitória. Indivíduos que apresentam doença periodontal representam possíveis reservatórios dessas bactérias oportunistas na cavidade bucal. O uso de antibióticos para o tratamento da doença periodontal ou outras infecções pode predispor o aumento do número de Staphylococcus spp. na boca, pois estes adquirem facilmente resistência aos antibióticos, podendo resultar em superinfecção. O objetivo deste estudo foi verificar a presença de Staphylococcus spp. na cavidade bucal e nas bolsas periodontais de pacientes com periodontite crônica; identificar as cepas isoladas; verificar a relação entre a presença de Staphylococcus spp. na cavidade bucal e presença de bolsa periodontal. Participaram deste estudo 88 pacientes, entre 25 e 60 anos de idade e apresentando periodontite crônica, com pelo menos dois sítios com profundidade de sondagem maior ou igual a 5mm. Após anamnese e exame clínico periodontal foram feitas coletas de material da bolsa periodontal com cones de papel e da cavidade bucal por meio de bochechos. Do total de pacientes 37,50% apresentaram Staphylococcus spp. na bolsa periodontal e 61,36% na cavidade bucal, sendo que 27,27% apresentaram a bactéria nos 2 sítios. S. epidermidis foi a espécie mais prevalente para bolsa periodontal (15,9%) e cavidade bucal (27,27%). Não houve diferença estatística significante quanto à presença desses microrganismos entre as faixas etárias e aumento da profundidade de sondagem. A presença de bactérias oportunistas na cavidade bucal pode representar dificuldades para a manutenção do tratamento periodontal. Palavras-chave: Staphylococcus , bactérias oportunistas, periodontite. INTRODUCTION Staphylococcus spp. display important virulence properties and cause a wide range of human infectious diseases including pneumonia, septicemia, and endocarditis (1,11,12,13,17,18, 21,22,23). They are not usually isolated from the oral cavity and when it occurs they are considered a part of the transient microbiota. However, a change in the oral microbiota may develop for several reasons. In immunocompromised individuals these microorganisms may occur in higher number. Patients with periodontal disease represent possible reservoirs of these opportunistic bacteria in the oral cavity. They can also be an infection source to other individuals (3,7,20). The purpose of this study was to evaluate the presence of Staphylococcus spp. in the oral cavity and in periodontal pockets of patients with chronic periodontitis, identify the isolates and verify the relationship between the presence of Staphylococcus spp. in the oral cavity and periodontal pocket. MATERIALS AND METHODS The study included 88 individuals with chronic periodontitis from the Clinic of Periodontology of the University of Taubaté (UNITAU). They were from 25 to 60 years of age and had not received periodontal nor antibiotic treatment. Following the examination, all patients were given instruction regarding to a proper self-performance plaque control. Prior to participation, the purpose and procedures were fully explained to all the patients. They volunteered themselves to participate in the trial and gave their informed consent (16). This study was carried out with the approval of the Ethics Committee of São José dos Campos Dentistry School (UNESP). Probing pocket depth and clinical attachment level were measured using a Williams' probe. Just one examiner, previously calibrated, examined all the patients. After individual data collection and clinical examination, samples of subgingival dental biofilm were obtained in those individuals that had at the time of referred at least two sites with probing depth > 5 mm. At the selected sites, supragengival plaque was removed with the aid of sterilized gauze compress and isolated with cotton rolls. Subgingival dental biofilm samples were collected by inserting 3 sterile paper points (number 30 Áureo) consecutively into the periodontal pocket, for 30 seconds. The collected material was placed in Eppendorf tubes containing 1mL of sterile solution (PBS 0.1M/pH 7.2). Material from oral cavity was collected through oral rinses, during 1min, in 10mL of sterile solution (PBS 0.1M/pH 7.2) previously distributed in sterile universal containers. The samples were maintained in ice until the process in the laboratory of Microbiology. The maximum period of 3 hours between collection and processing was respected. Microbiological analysis of the samples were performed in the laboratory of Microbiology, Department of Biology (UNITAU) or in the laboratory of Biosciences and Oral Diagnosis (São José dos Campos School of Dentistry, UNESP). The material obtained from periodontal pocket was mechanically dispersed with the aid of a Vortex mixer for 30 seconds. After this procedure, the paper points were removed.Each sample (oral rinse and periodontal pocket sample) was centrifuged by 10 min (8000 Xg) and the supernatant was discharged. The pellet was ressuspended in 2.5 mL and 0.6mL PBS respectively, obtaining the final suspension. From each sample 0.1 mL aliquots were plated in duplicate copy onto Baird-Parker agar (Difco) supplemented with egg yolk (12.5 egg yolks in 25ml saline solution o.85%) and potassium tellurite (0.1g of potassium tellurite in 10 mL of distilled water) and were incubated by 24 to 72 hours at 37ºC. After the incubation period, the identification of Staphylococcus isolates was based on colonial morphology and Gram stain characteristics. The strains were then on plated in Tryptic Soy Agar (TSA - Difco) and incubated by 24 hours at 37ºC. Pure cultures were identified according Koneman et al. (6). Coagulase negative Staphylococcus isolates were identified using the system API Staph (Bio-Merièux, France). The bacterial suspension preparation and the inoculation of the trips were carried out as recommended by the manufacturer. The strips were incubated by 24 hours at 37ºC. Based on the biochemical reactions, a seven-digit profile number was assigned to each isolate and the Code book were used for speciation. Chi-square homogeneity test and Chi-square tendency test were used with critical level at p< 0.05. RESULTS Out of the 88 patients with chronic periodontitis, staphylococci were isolated from the subgingival sample of 33 patients (37.50%); from the oral cavity of 54 patients (61.36%), and 27.27% (n=24) presented staphylococci in both sites. S. epidermidis (n=14, 15.90%) were isolated from the subgingival samples and 27.27% (n=24) from the oral cavity. Five patients (5.68%) present S. epidermidis in both sites. S. aureus were isolated from 4.55% of the subgingival samples and 25% (n=22) of the oral cavity samples ( Table 1 ). From the 88 studied patients, 39.77% were males (n=35) and 60.23% (n=53) females. The prevalence of Staphylococcus spp in the oral cavity of males was significantly higher in relation to the females. The prevalence of subgingival Staphylococcus according to the gender was not statistically significant ( Tables 2 and 3 ).The individuals ranged from 25 to 60 years of age (mean age 40.95)The presence of these microorganisms among the age groups was not statistically different ( Tables 4 and 5 ). In the present study, no significant correlation was detected between age and increase of the microorganisms ( Table 6 ). The higher number of positive samples for Staphylococcus spp was observed among the individuals between 31 and 40 years old (56,25%). For the probing depths between 5 to 6mm, 7 to 8mm and 9 to 10mm, the prevalence of S. epidermidis was of 15.15%, 17.07% and 14.29% respectively, for S. aureus was respectively of 6.06%, 0% and 14.29%. DISCUSSION Although staphylococci, mainly S. aureus and S. epidermidis, are frequently reported as pathogen causing nosocomial infections and acute problems, they are not frequently studied in the oral cavity. Subgingival staphylococci may not necessarily represent superinfection, but rather a colonization of indigenous oral bacteria. In the view of the virulence properties of these microorganisms, the present study examined the occurrence of these in oral cavity of patients with periodontal diseases. Slots et al. (19) found
Journal of Clinical Microbiology, Volume 40, pp 2936-2941; doi:10.1128/JCM.40.8.2936-2941.2002
The diagnosis of postsurgical mediastinitis (PSM) among patients with sternal wound complication (SWC) after cardiac surgery is sometimes difficult, as fever, elevated C-reactive protein levels, and chest pain can be caused by a general inflammatory reaction to the operative trauma and/or sternal dehiscence without infection. The definitions of PSM usually used emphasize clinical signs and symptoms easily observed by the surgeon. The aim of the study was to investigate whether the use of standardized multiple tissue sampling, optimal culturing methods, and strain typing, together with a microbiological criterion for infection, could identify more infected patients than clinical assessment alone. Patients reexplored due to SWC after cardiac artery bypass grafting (CABG) or heart valve replacement (HVR) with or without CABG performed at the Department for Cardio-Thoracic Surgery at the Uppsala University Hospital between 10 March 1998 and 9 September 2000 were investigated prospectively. Tissue samples were taken from the sternum or adjacent mediastinal tissue, preferably before the administration of antibiotics. Culturing was performed both directly (on agar plates) and using enrichment broth. Species identification was performed by standard methods, and strain typing was performed by pulsed-field gel electrophoresis. A total of 41 cases with at least five tissue samples each were included in the study group. Of these patients, 32 were infected according to the microbiological criterion (i.e., the same strain was found in ≥50% of the samples). Staphylococcus epidermidis was the primary pathogen in 38% of the cases (12/32), S. aureus was the primary pathogen in 31% (10/32), P. acnes was the primary pathogen in 25% (8/32), and S. simulans and S. haemolyticus were the primary pathogens in 3% (1/32) each. All cases of S. aureus infection and 86% (12/14) of coagulase-negative staphylococcus (CoNS) infections were identified from primary cultures. All cases fulfilling the microbiological criterion for S. aureus infection were clinically diagnosed as cases of infection, but among the 14 cases fulfilling the criterion for microbiological diagnosis of CoNS infection, only 10 appeared to qualify clinically as cases of infection. Among the patients with sternal dehiscence in whom a microbiological diagnosis was established, 67% (12/18) had a CoNS infection, compared to 14% (2/14) of those without sternal dehiscence. The difference was statistically significant. PSM caused by S. aureus is readily identified by the surgeon, whereas 30% of cases with CoNS infections may be misinterpreted as noninfected. Multiple sampling before administration of antibiotics, primary culturing on agar plates, species identification, strain typing, and susceptibility testing should be used to ensure a fast and microbiologically correct diagnosis which identifies the primary pathogen and infected patients among those with minor infective symptoms. The role of P. acnes as a...
Published: 1 August 2002
Journal of Clinical Microbiology, Volume 40
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